This invention relates to monoclonal antibodies to rapamycin and rapamycin derivatives, which are useful, e.g., in assay kits for monitoring blood levels of drug.
Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus, which has been found to be pharmaceutically useful in a variety of applications, particularly as an immunosuppressant, e.g., for use in the treatment and prevention of organ transplant rejection and autoimmune diseases. Rapamycin, however, does exhibit side effects at higher dosages, and it has a somewhat variable bioavailability. Monitoring blood levels of rapamycin in patients being treated with rapamycin is thus very desirable in order to be able to regulate the dosage so as to maintain the minimum level sufficient for pharmacologic activity and to avoid any undue risk of side effects. The lack of a sensitive and reliable assay which can be performed quickly and easily in a clinical setting has been a major obstacle to the development of rapamycin as a pharmaceutical.
Previous efforts to develop assay kits for clinical monitoring of rapamycin have not been particularly successful. EP 041795, for example, describes a microbiological assay in which rapamycin concentration is measured as a function of antifungal activity. WO 92/02946 provides an assay system which measures rapamycin levels indirectly by measuring competition for binding to macrophilin. Both of these assays are cumbersome and not particularly sensitive. Even more importantly, both of these assays may have considerable variation under slightly different test conditions, making comparisons of test results from different hospitals difficult.
There have been no previous reports of monoclonal antibodies which recognize rapamycin. There are inherent difficulties in making monoclonal antibodies to rapamycin because rapamycin is not immunogenic and is itself extremely immunosuppressive. Moreover, as the metabolites of rapamycin have not been well characterized in the literature, it is difficult to identify a monoclonal antibody capable of differentiating between rapamycin and its metabolites.
The present invention provides monoclonal antibodies which are highly sensitive to rapamycin. The antibodies of the invention are produced in response to inoculation with a novel immunogenic conjugate comprising a novel derivative of rapamycin linked to an immunogenic protein. Assay kits using these antibodies are well suited for use in a clinical setting and provide far more accurate and reproducible results than was previously possible. The antibodies are also useful in the purification and isolation of rapamycin.
Providing assay systems for immunosuppressive derivatives of rapamycin present similar challenges. Of particular interest are 40-O-derivatives of rapamycin, i.e., rapamycins which are O-substituted at the hydroxy on the cyclohexyl ring (position 40), e.g., as described in U.S. Pat. No. 5,258,389 and PCT/EP 93/02604 (O-aryl and O-alkyl rapamycins) (both incorporated herein by reference); especially 40-O-alkylated rapamycins where the 40-O-substituent is alkyl or substituted alkyl; e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, wherein xe2x80x9calk-xe2x80x9d or xe2x80x9calkylxe2x80x9d refers to C1-6 alkyl, branched or linear, preferably C1-3 alkyl, in which the carbon chain may be optionally interrupted by an ether (xe2x80x94Oxe2x80x94) linkage; most especially 40-O-(2-hydroxyethyl)-rapamycin, 40-O-(3-hydroxypropyl)-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and 40-O-(2-acetaminoethyl)-rapamycin). Thus a further object of the invention is to provide monoclonal antibodies to such 40-O-derivatives. Such antibodies are useful in diagnostic assays and also in the purification and production of the derivatives.
The novel activated derivatives of rapamycin used to make the novel immunogenic conjugates of the invention are rapamycins which are linked through one of the hydroxy groups on the rapamycin, preferably the hydroxy group located on the cyclohexyl portion of the rapamycin (position 40) or the hydroxy at position 28, to an activated coupling group, i.e., a group capable of direct reaction with a protein to form a covalent linkage without the requirement for the use of a coupling agent (e.g., carbodiimide reagents) to enable, effect, or promote the reaction with the protein. Preferably, the activated coupling group has an activated ester or carboxy group, i.e., of formula xe2x80x94COxe2x80x94Oxe2x80x94X where X is a carboxy activating group such as o- or p-nitrophenyl, 1-benztriazole, pentafluorophenyl, or (especially) N-succinimido. Other suitable activated coupling groups are, for example, i) activated dithio groups, e.g., of formula xe2x80x94Sxe2x80x94Sxe2x80x94Z wherein Z is a dithio activating group such as 2-pyridyl, which may be linked to the rapamycin; or ii) epoxy groups, e.g., epoxy methyl. The activated coupling group may be linked to the rapamycin by means of an ester, ether, amide, thio or other suitable linkage, but ester linkage is preferred. Most preferably, the activated coupling group contains a bis-ester moiety, e.g., succinyl, having an ester linkage to the rapamycin at one end and the activated ester or activated carboxy group at the other.
The preferred rapamycin derivatives of the invention are those of formula III below which are produced according to Reaction I: 
wherein formula I is rapamycin, which is a) reacted with an acylating agent, e.g., a cyclic anhydride or a dicarboxylic acid (optionally in hemi-O-protected form), under suitable conditions and deprotection if necessary to yield the rapamycin of formula II, wherein Y is a spacer moiety, preferably a lower alkylene, e.g., C2-6 alkylene, most preferably ethylene. This rapamycin of formula II is then b) activated by reaction with a carboxy activating group, e.g. of formula HOxe2x80x94X where X is as defined above, to yield the activated rapamycin of formula III.
A preferred activated derivative of rapamycin is the succinimido derivative of formula III below, prepared, e.g., according to Reaction II: 
wherein formula I is rapamycin, which is a) 0-acylated using succinic anhydride in the presence of DMAP and pyridine to form the rapamycin hemisuccinate of formula IIxe2x80x2 (40-O-(3-Carboxy)propanoyl-rapamycin); which is then b) activated with N-Hydroxy succinimide in the presence of EDC, Et3N, and CH2Cl2 to form the 40-O-succinimidooxysuccinyl rapamycin of formula IIIxe2x80x2, e.g., as described more fully in example 1 below. Monoclonal antibodies produced using a hapten such as this which is linked through the 40-position will ordinarily be cross reactive between rapamycin and a 40-O-derivative of rapamycin, such as described above. Such monoclonal antibodies can be selected as described below for compounds which recognize a particular region of the rapamycin or 40-O-derivative of the rapamycin, e.g., in the binder domain or effector domain, as described below.
It is in some cases desirable to have monoclonal antibodies capable of fine sensitivity to modifications in the cyclohexyl region, e.g., for distinguishing between rapamycin and the 40-O rapamycin derivatives, or for identifying metabolites in the cyclohexyl region. In such a case, the hapten is preferably linked through the 28-O position rather then the 40-O position. For example, the rapamycin derivative of formula A: 
wherein R is an O-protecting group, or a substituent as described above, e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, optionally in protected form, is reacted according to Reaction I, deprotecting if necessary, to give the analogous 28-O activated hapten, for example a compound of formula B: 
wherein R1 is H, or an O-substituent as described above, e.g., hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, Y is a linker moiety as defined above, and X is a carboxy activating group as defined above. In preparing this hapten, where R is an O-protecting group or an O-protected substituent, the acylating agent may optionally be, e.g., a dicarboxylic acid in hemi-O-protected form, so that following acylation, both O-protecting groups may be removed in one step prior to adding the carboxy activating group. For example, haptens for generating monoclonal antibodies capable of recognizing 40-O-(2-hydroxyethyl)-rapamycin, can be produced by protecting the primary hydroxy, acylating the hydroxy at position 28 with a dicarboxylic acid in hemi-O-protected form, deprotecting, and activating the carboxy group, e.g., according to Reaction III: 
Similarly, rapamycin itself may be activated at the 28-O rather than the 40-O, by O-protecting the C40 hydroxy, acylating the hydroxy group in position 28 with a hemi-O-protected dicarboxylic acid, deprotecting, and activating the carboxy group, e.g., according reaction IV: 
The activated rapamycin or rapamycin derivative is then linked to a suitable immunogenic protein, e.g., bovine serum albumin (BSA), ovalbumin (OVA), or keyhole limpet hemocyanine (KLH) to form an immunogenic conjugate. Monoclonal antibodies are prepared using conventional methods, e.g., administering the novel immunogenic conjugate to a suitable animal species to effect immunogenic challenge and recovering antibody-producing cells sensitized to said conjugate; immortalizing said antibody producing cells by fusion with a suitable myeloma; and recovering the monoclonal antibody from a selected immortalized cell line thus established.
The antibodies of the invention may then be used in a suitable assay. Several possibilities would be clear to one skilled in the art. One approach is a competitive assay using antibody and a rapamycin tracer, for example wherein microtiter plates are coated with antibody and exposed to a competitor which is a labeled (e.g., fluoro- or radio-labeled, especially biotinylated) rapamycin, in the presence and absence of test fluid believed to possibly contain a rapamycin, e.g., plasma or whole blood from the patient. The plates are rinsed, and the amount of labeled competitor which has bound to the antibody is measured, which amount varies inversely with the amount of rapamycin in the test fluid. Another approach is an ELISA using antibody, a rapamycin protein conjugate, and a labeled (e.g., enzyme-labeled) tracer antibody recognizing murine IgG, for example wherein microtiter plates are coated with a rapamycin-protein conjugate (e.g., the immunogenic conjugate described above comprising a protein linked to rapamycin or a 40-O-alkylated rapamycin), exposed to antibody in the presence and absence of test fluid, rinsed, and antibody binding to the rapamycin conjugate detected by binding of the tracer antibody to the antibody bound to the rapamycin conjugate. Again, the amount of bound antibody will vary inversely with the amount of rapamycin in the test sample. In either case, the assay is standardized with test solutions containing known concentrations of rapamycin. An assay kit comprising (i) the monoclonal antibody of the invention, preferably in lyophilized form or coated onto a microtiter plate, and (ii) optionally also comprising either a rapamycin protein conjugate, optionally coated onto a plate, and/or a labeled rapamycin derivative, and (iii) further optionally comprising a rapamycin solution for standardization and instructions for use, is therefore provided. Such a kit is capable of detecting rapamycin at concentrations of below 10 ng/ml, e.g., below 1 ng/ml, e.g., as low as 0.25-0.5 ng/ml.
The antibodies of the invention may be further characterized by their relative binding affinity to an immunosuppressive ascomycin, e.g., FK-506. FK-506 is an immunosuppressive macrolide having some structural similarity to rapamycin in the binding domain. Rapamycins (e.g., rapamycin and its immunosuppressant derivatives) and FK-506 both bind to macrophilins (FKBPs), and for both it is believed that macrophilin binding is a necessary but not a sufficient criteria for immunosuppressive activity. The effector region of rapamycin, however, is quite different from FK-506, and indeed, the two compounds have quite different mechanisms of activity. (FK-506 for example appears to cause immunosuppression primarily by suppressing IL-2 transcription, whereas rapamycin has no significant effect on IL-2 transcription.) Rapamycins can thus be characterized as having an FKBP binding domain and an effector domain, and a distinction can be made between rapamycin metabolites which are modified in the FKBP binding domain from those modified in the effector domain. This distinction can be made with the monoclonal antibodies of the invention by measuring the relative cross-reactivity of the monoclonal antibodies of the invention with FK-506 (cross-reactivity being measured, e.g., in a competitive ELISA): monoclonal antibodies having a high degree of cross reactivity (e.g., greater than 50%) recognize epitopes in the FKBP binding domain of rapamycin which is similar to FK-506; monoclonal antibodies with a low degree of cross reactivity (e.g., less than 20%, optimally less than 10%) recognize epitopes in the effector region, which is unique to rapamycins.
Antibodies of the invention can also be screened and characterized according to their ability to distinguish between rapamycin and a 40-O-derivative of rapamycin, e.g., as defined above. Where it is desired that the antibodies do not distinguish between rapamycin and a 40-O-derivative of rapamycin, antibodies are selected which show at least 70%, preferably greater than 90%, cross-reactivity between rapamycin and a 40-O-derivative thereof. In such a case, the hapten used to make the monoclonal antibody is preferably a 40-O-activated rapamycin, e.g., of formula III in Reaction I. Where it is desired to distinguish between rapamycin and a 40-O-derivative or metabolite of rapamycin, antibodies are selected having less than 30%, preferably less than 10%, cross-reactivity thereto. In this case, the hapten used to make the antibody is preferably a 28-O-activated rapamycin or rapamycin derivative, e.g., of formula B.